Methods and compositions to evaluate antibody treatment response

ABSTRACT

The present invention relates to methods and compositions to evaluate or assess the response of a subject to particular therapeutic treatment. More particularly, the invention provides methods to determine the response of subjects, or to adapt the treatment protocol of subjects treated with therapeutic antibodies. The invention is based on a determination of the FCGR3A genotype of a subject. The invention can be used for patients with malignancies, particularly lymphoma, and is suited to select best responders and/or adjust treatment condition or protocol for low responders.

The present invention relates to methods and compositions to evaluate orassess the response of a subject to particular therapeutic treatment.More particularly, the invention provides methods to determine theresponse of subjects, or to adapt the treatment protocol of subjectstreated with therapeutic antibodies. The invention can be used forpatients with malignancies, particularly lymphoma, and is suited toselect best responders and/or adjust treatment condition or protocol forlow responders.

INTRODUCTION

Various therapeutic strategies in human beings are based on the use oftherapeutic antibodies. This includes, for instance, the use oftherapeutic antibodies developed to deplete target cells, particularlydiseased cells such as virally-infected cells, tumor cells or otherpathogenic cells, including allogenic immunocompetent cells. Suchantibodies are typically monoclonal antibodies, of IgG species,typically IgG1 and IgG3. These antibodies can be recombinant antibodiesand humanized antibodies, comprising functional domains from variousspecies or origin or specificity. A particular example of suchtherapeutic antibodies is rituximab (Mabthera®, Rituxan®), which is achimeric anti-CD20 IgG1 monoclonal antibody made with human γ1 and κconstant regions linked to murine variable domains¹. For a few years,rituximab has been considerably modifying the therapeutical strategyagainst B lymphoproliferative malignancies, particularly non-Hodgkin'slymphomas (NHL). Other examples of intact humanized IgG1 antibodiesinclude alemtuzumab (Campath-1H®), which is used in the treatment of Bcell malignancies or trastuzumab (Herceptin®, which is used in thetreatment of breast cancer. Additional examples of therapeuticantibodies under development are disclosed in the art.

While these antibodies represent a novel efficient approach to humantherapy, particularly for treatment of tumors, they do not alwaysexhibit a strong efficacy and their use could be improved by evaluatingthe response of subjects thereto. For instance, while rituximab, aloneor in combination with chemotherapy was shown to be effective in thetreatment of both low-intermediate²⁻⁸ and high-grade NHL^(6, 9), 30% to50% of patients with low grade NHL have no clinical response torituximab^(4, 5). It has been suggested that the level of CD20expression on lymphoma cells², the presence of high tumor burden at thetime of treatment⁶ or low serum rituximab concentrations² may explainthe lack of efficacy of rituximab in some patients. Nevertheless, theactual causes of treatment failure remain largely unknown.

The availability of methods allowing the evaluation of patient responseto antibody treatment would greatly enhance the therapeutic efficacy ofthese products. However, the precise mode of action in vivo of suchtherapeutic antibodies is not clearly documented. Indeed, while in vitrostudies suggest various possible modes of action of rituximab(antibody-dependant cell-mediated cytotoxicity (ADCC)^(10, 11),complement-dependant cytotoxicity^(10, 12, 13), direct signallingleading to apoptosis^(14, 15), etc.), the clear action of these targetcell-depleting antibodies in vivo is not documented in humans.Furthermore, while ADCC is an important effector mechanism in theeradication of intracellular pathogens and tumor cells, the role of anADCC is still controversial^(12, 13).

The present invention now proposes novel methods and compositions toassess the therapeutic response of a subject to a therapeutic antibody.The invention also proposes methods to select patients having bestresponding profile to therapeutic antibody treatment. The invention alsorelates to methods of treating patients with therapeutic antibodies,comprising a prior step of evaluating the patient's response. Theinvention also relates to compositions and kits suitable to perform theinvention. The invention may as well be used in clinical trials orexperimental settings, to assess or monitor a subject's response, or toverify the mode of action of an antibody.

The invention is based, in part, on the demonstration of a correlationbetween the genotype of a subject and its ability to respond totherapeutic antibody treatment. More specifically, the invention showsthat the genotype of the FcγRIIIa receptor directly correlates with thesubject's response to therapeutic antibody treatment.

Three classes of FcγR (FcγRJ, FcγRII and FcγRIII) and their subclassesare encoded by eight genes in humans, all located on the long arm ofchromosome 1. Some of these genes display a functional allelicpolymorphism generating allotypes with different receptor properties.These polymorphisms have been identified as genetic factors increasingthe susceptibility to autoimmune or infectious diseases¹⁹⁻²¹. One ofthese genetic factors is a gene dimorphism in FCGR3A, which encodesFcγRIIIa with either a phenylalanine (F) or a valine (V) at amino-acidposition 158^(22,23). This residue directly interacts with the lowerhinge region of IgG1 as recently shown by IgG1-FcγRIIIco-cristallization²⁴. It has been clearly demonstrated that human IgG1binds more strongly to homozygous FcγRIIIa-158V natural killer cells(NK) than to homozygous FcγRIIIa-158F or heterozygous NK cells^(22, 23).

We undertook to evaluate a possible correlation between the FCGR3Agenotype and a patient response to therapeutic antibody treatment invivo. Our invention stems in part from the unexpected discovery that avery strong correlation exists between said genotype and said responseprofile, the presence of a valine residue at position 158 beingindicative of a high response rate. More specifically, the genotyping ofFCGR3A was performed in patients with previously untreated follicularNHL who had received rituximab alone, a particular situation in whichthe response rate is very high⁵. The FCGR2A-131H/R was also determinedas control since this gene co-localizes with FCGR3A on chromosome 1q22and encodes the macrophage FcγRIIa receptor.

The FCGR3A-158V/F genotype was determined in 47 patients having receivedrituximab for a previously untreated follicular non-Hodgkin's lymphoma.Clinical and molecular response were evaluated at two months (M2) and atone year (M12). Positive molecular response was defined as adisappearance of the BCL2-JH gene rearrangement in both peripheral bloodand bone marrow. FCGR3A-158V homozygous patients were 21% whereasFCGR3A-158F homozygous and heterozygous patients (FCGR3A-158F carriers)were 34% and 45%, respectively. The objective response rates at M2 andM12 were 100% and 90% in FCGR3A-158V homozygous patients compared with65% (p=0.02) and 51% (p=0.03) in FCGR3A-158F carriers. A positivemolecular response was observed at M12 in 5/6 of homozygous FCGR3A-158Vpatients compared with 5/16 of FCGR3A-158F carriers (p=0.04).Furthermore, the homozygous FCGR3A-158V genotype was confirmed to be thesingle parameters associated with clinical and molecular responses inmultivariate analysis and was also associated with a lower rate ofdisease progression (p=0.05).

Accordingly, the present invention establishes, for the first time, anassociation between the FCGR3A genotype and clinical and molecularresponses to therapeutic antibodies. The invention thus provides a firstunique marker that can be used to monitor, evaluate or select apatient's response. This invention thus introduces new pharmacogeneticalapproaches in the management of patients with malignancies, viralinfections or other diseases related to the presence of pathologicalcells in a subject, particularly non-Hodgkin's lymphoma.

An object of this invention resides in a method of assessing theresponse of a subject to a therapeutic antibody treatment, comprisingdetermining in vitro the FCGR3A genotype and/or the presence of apolymorphism in the FcγRIIIa receptor of said subject. Morespecifically, the method comprises determining in vitro the FCGR3A158genotype of said subject.

A further object of this invention is a method of selecting patients fortherapeutic antibody treatment, the method comprising determining invitro the FCGR3A genotype and/or the presence of a polymorphism in theFcγRIIIa receptor of said subject. More specifically, the methodcomprises determining in vitro the FCGR3A 158 genotype of said subject.

An other object of this invention is a method of improving the efficacyor treatment condition or protocol of a therapeutic antibody treatmentin a subject, comprising determining in vitro the FCGR3A genotype and/orthe presence of a polymorphism in the FcγRIIIa receptor of said subject.More specifically, the method comprises determining in vitro the FCGR3A158 genotype of said subject.

More specifically, determining in vitro the FCGR3A 158 genotype of asubject comprises determining amino acid residue at position 158 ofFcγRIIIa receptor (or corresponding codon in the FCGR3A gene), a valineat position 158 being indicative of a better response to said treatmentand a phenylalanine at position 158 being indicative of a lower responseto said treatment.

Within the context of this invention, the term “therapeutic antibody orantibodies” designates more specifically any antibody that functions todeplete target cells in a patient. Specific examples of such targetcells include tumor cells, virus-infected cells, allogenic cells,pathological immunocompetent cells (e.g., B lymphocytes, T lymphocytes,antigen-presenting cells, etc.) involved in allergies, autoimmunediseases, allogenic reactions, etc., or even healthy cells (e.g.,endothelial cells in an anti-angiogenic therapeutic strategy). Mostpreferred target cells within the context of this invention are tumorcells and virus-infected cells. The therapeutic antibodies may, forinstance, mediate a cytotoxic effect or a cell lysis, particularly byantibody-dependant cell-mediated cytotoxicity (ADCC). ADCC requiresleukocyte receptors for the Fc portion of IgG (FcγR) whose function isto link the IgG-sensitized antigens to FcγR-bearing cytotoxic cells andto trigger the cell activation machinery. While this mechanism of actionhas not been evidenced in vivo in humans, it may account for theefficacy of such target cell-depleting therapeutic antibodies. Thetherapeutic antibodies may by polyclonal or, preferably, monoclonal.They may be produced by hybridomas or by recombinant cells engineered toexpress the desired variable and constant domains. The antibodies may bysingle chain antibodies or other antibody derivatives retaining theantigen specificity and the lower hinge region or a variant thereof.These may be polyfunctional antibodies, recombinant antibodies, ScFv,humanized antibodies, or variants thereof. Therapeutic antibodies arespecific for surface antigens, e.g., membrane antigens. Most preferredtherapeutic antibodies are specific for tumor antigens (e.g., moleculesspecifically expressed by tumor cells), such as CD20, CD52, ErbB2 (orHER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, αVβ3, etc., particularlylymphoma antigens (e.g., CD20). The therapeutic antibodies arepreferably IgG1 or IgG3, more preferably IgG1.

Typical examples of therapeutic antibodies of this invention arerituximab, alemtuzumab and trastuzumab. Such antibodies may be usedaccording to clinical protocols that have been authorized for use inhuman subjects. Additional specific examples of therapeutic antibodiesinclude, for instance, epratuzumab, basiliximab, daclizumab, cetuximab,labetuzumab, sevirumab, tuvurimab, palivizumab, infliximab, omalizumab,efalizumab, natalizumab, clenoliximab, etc., as listed in the followingtable:

Ab specificity DCI Commercial name Typical Indications Anti-CD20rituximab MabThera ®, LNH B Rituxan ® Anti-CD52 alemtuzumab CAMPATH-1H ®LLC, allograft Anti-CD33 Zamyl ™ Acute myeloid Leukemia Anti-HLA-Remitogen ™ LNH B DR Anti-CD22 epratuzumab LymphoCide ™ LNH B Anti-erbB2trastuzumab Herceptin ®, Metastatic breast cancer (HER-2/neu) Anti-EGFRcetuximab ORL and colorectal Cancers (HER-1, erbB1) Anti-MUC-1 Therex ®Breast and epithelial cancers Anti-CEA labetuzumab CEA-Cide ™ Anti-αVβ3Vitaxin Cancers (anti-angiogenic) Anti-KDR Cancers (anti-angiogenic)(VEGFR2) anti-VRS palivizumab Synagis ® Viral diseases fusion proteinanti-VRS Numax ™ ″ fusion protein CMV sevirumab Protovir CMV InfectionHBs tuvirumab Ostavir ™ Hepatitis B Anti-CD25 basiliximab Simulect ®Prevention/treatment allograft rejection Anti-CD25 daclizumab Zénapax ®Prevention/treatment allograft rejection anti-TNF-α infliximabRemicade ™ Crohn disease, polyarthrite rhumatoïd anti-IgE omalizumabXolair ™ Asthma anti-integrin efalizumab Xanelim ™ psoriasis αL (CD11a,LFA-1) anti-CD4 keliximab anti-CD2 siplizumab Anti-CD64 anemiaanti-CD147 GvH anti-integrin natalizumab Antegren ® Sclerosis, Crohn α4(α4β1-α4β7) Anti-integrin Crohn, RCH β7 anti-CD4* clenoliximab

Within the context of the present invention, a subject or patientincludes any mammalian subject or patient, more preferably a humansubject or patient.

According to the invention the term FCGR3A gene refers to any nucleicacid molecule encoding a FcγRIIIa polypeptide in a subject. This termincludes, in particular, genomic DNA, cDNA, RNA (pre-rRNA, messengerRNA, etc.), etc. or any synthetic nucleic acid comprising all or part ofthe sequence thereof. Synthetic nucleic acid includes cDNA, preparedfrom RNAs, and containing at least a portion of a sequence of the FCGR3Agenomic DNA as for example one or more introns or a portion containingone or more mutations. Most preferably, the term FCGR3A gene refers togenomic DNA, cDNA or mRNA, typically genomic DNA or mRNA. The FCGR3Agene is preferably a human FCGRIIIa gene or nucleic acid, i.e.,comprises the sequence of a nucleic acid encoding all or part of aFcγRIIIa polypeptide having the sequence of human FcγRIIIa polypeptide.Such nucleic acids can be isolated or prepared according to knowntechniques. For instance, they may be isolated from gene libraries orbanks, by hybridization techniques. They can also be genetically orchemically synthesized. The genetic organization of a human FCGRIIIagene is depicted on FIG. 2. The amino acid sequence of human FcγRIIIa isrepresented FIG. 3. Amino acid position 158 is numbered from residue 1of the mature protein. It corresponds to residue 176 of the pre-proteinhaving a signal peptide. The sequence of a wild type FCGR3A gene isrepresented on FIG. 4 (see also Genbank accession Number AL590385 orNM_(—)000569 for partial sequence).

Within the context of this invention, a portion or part means at least 3nucleotides (e.g., a codon), preferably at least 9 nucleotides, evenmore preferably at least 15 nucleotides, and can contain as much as 1000nucleotides. Such a portion can be obtained by any technique well knownin the art, e.g., enzymatic and/or chemical cleavage, chemical synthesisor a combination thereof. The sequence of a portion of a FCGR3A geneencoding amino acid position 158 is represented below, for sake ofclarity:

cDNA 540       550       560       570       580 genomic DNA   4970      4980      4990      5000. 158F alleletcctacttctgcagggggctttttgggagtaaaaatgtgtcttca  S  Y  F  C  R  G  L   F  G  S  K  N  V  S  S 158V alleletcctacttctgcagggggcttgttgggagtaaaaatgtgtcttca  S  Y  F  C  R  G  L   V  G  S  K  N  V  S  S

As indicated above, the invention comprises a method of determining invitro the FCGR3A158 genotype of said subject. This more particularlycomprises determining the nature of amino acid residue present (orencoded) at position 158 of the FcγRIIIa polypeptide.

Genotyping the FCGR3A gene or corresponding polypeptide in said subjectmay be achieved by various techniques, comprising analysing the codingnucleic acid molecules or the encoded polypeptide. Analysis may comprisesequencing, migration, electrophoresis, immuno-techniques,amplifications, specific digestions or hybridisations, etc.

In a particular embodiment, determining amino acid residue at position158 of FcγRIIIa receptor comprises a step of sequencing the FCGR3Areceptor gene or RNA or a portion thereof comprising the nucleotidesencoding amino acid residue 158.

In an other particular embodiment, determining amino acid residue atposition 158 of FcγRIIIa receptor comprises a step of amplifying theFCGR3A receptor gene or RNA or a portion thereof comprising thenucleotides encoding amino acid residue 158. Amplification may beperformed by polymerase chain reaction (PCR), such as simple PCR, RT-PCRor nested PCR, for instance, using conventional methods and primers.

In this regard, amplification primers for use in this invention morepreferably contain less than about 50 nucleotides even more preferablyless than 30 nucleotides, typically less than about 25 or 20nucleotides. Also, preferred primers usually contain at least 5,preferably at least 8 nucleotides, to ensure specificity. The sequenceof the primer can be prepared based on the sequence of the FCGR3A gene,to allow full complementarity therewith, preferably. The probe may belabelled using any known techniques such as radioactivity, fluorescence,enzymatic, chemical, etc. This labeling can use for example Phosphor 32,biotin (16-dUTP), digoxygenin (11-dUTP). It should be understood thatthe present invention shall not be bound or limited by particulardetection or labelling techniques. The primers may further compriserestriction sites to introduce allele-specific restriction sites in theamplified nucleic acids, as disclosed below.

Specific examples of such amplification primers are, for instance, SEQID NO: 1-4.

It should be understood that other primers can be designed by theskilled artisan, such as any fragment of the FCGR3A gene, for use in theamplification step and especially a pair of primers comprising a forwardsequence and a reverse sequence wherein said primers of said pairhybridize with a region of a FCGR3A gene and allow amplification of atleast a portion of the FCGR3A gene containing codon 158. In a preferredembodiment, each pair of primers comprises at least one primer that iscomplementary, and overlaps with codon 158, and allows to discriminatebetween 158V (gtt) and 158F (ttt). The amplification conditions may alsobe adjusted by the skilled person, based on common general knowledge andthe guidance contained in the specification.

In a particular embodiment, the method of the present invention thuscomprises a PCR amplification of a portion of the FCGR3a mRNA or gDNAwith specific oligonucleotide primers, in the cell or in the biologicalsample, said portion comprising codon 158, and a direct or indirectanalysis of PCR products, e.g., by electrophoresis, particularlyDenaturing Gel Gradient Electrophoresis (DGGE).

In an other particular embodiment, determining amino acid residue atposition 158 of FcγRIIIa receptor comprises a step of allele-specificrestriction enzyme digestion. This can be done by using restrictionenzymes that cleave the coding sequence of a particular allele (e.g.,the 158V allele) and that do not cleave the other allele (e.g., the 158Fallele, or vice versa). Where such allele-specific restriction enzymesites are not present naturally in the sequence, they may be introducedtherein artificially, by amplifying the nucleic acid withallele-specific amplification primers containing such a site in theirsequence. Upon amplification, determining the presence of an allele maybe carried out by analyzing the digestion products, for instance byelectrophoresis. This technique also allows to discriminate subjectsthat are homozygous or heterozygous for the selected allele.

Examples of allele-specific amplification primers include for instanceSEQ ID NO: 3. SEQ ID NO:3 introduces the first 3 nucleotides of theNlaIII site (5′-CATG-3). Cleavage occurs after G. This primer comprises11 bases that do not hybridise with FCGR3A, that extend the primer inorder to facilitate electrophoretic analysis of the amplificationproducts) and 21 bases that hybridise to FCGR3A, except for nucleotide31 (A) which creates the restriction site.

In a further particular embodiment, determining amino acid residue atposition 158 of FcγRIIIa receptor comprises a step of hybridization ofthe FCGR3A receptor gene or RNA or a portion thereof comprising thenucleotides encoding amino acid residue 158, with a nucleic acid probespecific for the genotype Valine or Phenylalanine, and determining thepresence or absence of hybrids.

It should be understood that the above methods can be used either aloneor in various combinations. Furthermore, other techniques known to theskilled person may be used as well to determine the FCGR3A158 genotype,such as any method employing amplification (e.g. PCR), specific primers,specific probes, migration, etc., typically quantitative RT-PCR, LCR(Ligase Chain Reaction), TMA (Transcription Mediated Amplification), PCE(an enzyme amplified immunoassay) and bDNA (branched DNA signalamplification) assays.

In a preferred embodiment of this invention, determining amino acidresidue at position 158 of FcγRIIIa receptor comprises:

-   -   Obtaining genomic DNA from a biological sample,    -   Amplifying the FcγRIIIa receptor gene or a portion thereof        comprising the nucleotides encoding amino acid residue 158, and    -   determining amino acid residue at position 158 of said FcγRIIIa        receptor gene.

Amplification can be accomplished with any specific technique such asPCR, including nested PCR, using specific primers as described above. Ina most preferred embodiment, determining amino acid residue at position158 is performed by allele-specific restriction enzyme digestion. Inthat case, the method comprises:

-   -   Obtaining genomic DNA from a biological sample,    -   Amplifying the FcγRIIIa receptor gene or a portion thereof        comprising the nucleotides encoding amino acid residue 158,    -   Introducing an allele-specific restriction site,    -   Digesting the nucleic acids with the enzyme specific for said        restriction site and,    -   Analysing the digestion products, i.e., by electrophoresis, the        presence of digestion products being indicative of the presence        of the allele.

In an other particular embodiment, the genotype is determined by amethod comprising: total (or messenger) RNA extraction from cell orbiological sample or biological fluid in vitro or ex vivo, optionallycDNA synthesis, (PCR) amplification with FCGR3A-specific oligonucleotideprimers, and analysis of PCR products.

The method of this invention may also comprise determining amino acidresidue at position 158 of FcγRIIIa receptor directly by sequencing theFcγRIIIa receptor polypeptide or a portion thereof comprising amino acidresidue 158 or by using reagents specific for each allele of theFcγRIIIa polypeptide. This can be determined by any suitable techniqueknown to the skilled artisan, including by immuno-assay (ELISA, EIA,RIA, etc.). This can be made using any affinity reagent specific for aFcγRIIIa158 polypeptide, more preferably any antibody or fragment orderivative thereof. In a particular embodiment, the FcγRIIIa158polypeptide is detected with an anti-FcγRIIIa158 antibody (or a fragmentthereof) that discriminates between FcγRIIIa158V and FcγRIIIa158F, morepreferably a monoclonal antibody. The antibody (or affinity reagent) maybe labelled by any suitable method (radioactivity, fluorescence,enzymatic, chemical, etc.). Alternatively, FcγRIIIa158 antibody immunecomplexes may be revealed (and/or quantified) using a second reagent(e.g., antibody), labelled, that binds to the anti-FcγRIIIa158 antibody,for instance.

The above methods are based on the genotyping of FCGR3A158 in abiological sample of the subject. The biological sample may be anysample containing a FCGR3A gene or corresponding polypeptide,particularly blood, bone marrow, lymph node or a fluid, particularlyblood or urine, that contains a FCGR3A158 gene or polypeptide.Furthermore, because the FCGR3A 158 gene is generally present within thecells, tissues or fluids mentioned above, the method of this inventionusually uses a sample treated to render the gene or polypeptideavailable for detection or analysis. Treatment may comprise anyconventional fixation techniques, cell lysis (mechanical or chemical orphysical), or any other conventional method used in immunohistology orbiology, for instance.

The method is particularly suited to determine the response of a subjectto an anti-tumor therapeutic antibody treatment. In this regard, in aparticular embodiment, the subject has a tumor and the therapeuticantibody treatment aims at reducing the tumor burden, particularly atdepleting the tumor cells. More preferably, the tumor is a lymphoma,such as more preferably a B lymphoma, particularly a NHL. As indicatedabove, the antibody is preferably an IgG1 or an IgG3, particularly ananti-CD20 IgG1 or IgG3, further preferably a humanized antibody, forinstance rituximab.

The invention also relates to a bispecific antibody, wherein saidbispecific antibody specifically binds CD16 and a tumor antigen, forinstance a CD20 antigen. The invention also encompasses pharmaceuticalcompositions comprising such a bispecific antibody and apharmaceutically acceptable excipient or adjuvant.

Further aspects and advantages of this invention will be disclosed inthe following examples, which should be regarded as illustrative and notlimiting the scope of this application.

FIGURE LEGENDS

FIG. 1: Adjusted KAPLAN-METER estimates of progression-free survivalafter rituximab treatment according to FcγR3a-158V/F genotype (p=0.05).

FIG. 2: Genetic organization of the human FCGR3A gene

FIG. 3: Amino acid sequences of human FcγRIIIa158F (SEQ ID NO:7)

FIG. 4: Nucleic acid sequence of human FCGR3A158F (SEQ ID NO:8)

MATERIALS AND METHODS Patients and Treatment

Clinical trial design, eligibility criteria and end-point assessmenthave been previously reported.⁵ In brief, patients were eligible forinclusion in this study if they had previously untreated follicular CD20positive NHL according to the REAL classification.²⁶ Patients wererequired to present with stage II to IV disease according to Ann-Arborclassification and at least one measurable disease site. All patientswere required to have low tumor burden according to the GELF criteria.²⁷A total of four 375 mg/m² doses of rituximab (Roche, Neuilly, France)were administered by intravenous infusion (days 1, 8, 15, 22). Themanagement of infusion and adverse events has already been reported.⁵The study protocol was approved by an ethics committee, and all patientsgave their informed consent.

Monitoring and Endpoints

Baseline evaluation included clinical examination, chest X-ray, computedtomography (CT) of the chest, abdomen and pelvis, and unilateral bonemarrow biopsy. Response was assessed by an independent panel ofradiologists who reviewed all the CT scans of the included patients. Theprimary efficacy endpoint was the objective response rate, i.e theproportion of patients achieving either complete remission (CR),unconfirmed CR (CRu) or partial response (PR) according to the criteriarecently proposed by an international expert committee.²⁸ Clinicalresponse was evaluated at days 50 and 78. Only the maximum response wastaken into account and that assessment time point named M2. All patientswere evaluated for progression at one year (M12). Patients in CR or CRuwith disappearance of bone marrow infiltration at M2 and reappearance oflymphoma cells in bone marrow at M12 were considered “progressive”;patients in PR with negative bone marrow biopsy at M2 and positivebiopsy at M12 were considered in PR.

Molecular analysis of BCL2-JH gene rearrangement was performed by PCR,as previously described,⁵ on a lymph node obtained at diagnosis and onboth peripheral blood and bone marrow at diagnosis, M2 and M12.

FCGR3A-158V/F Genotyping

Out of the 50 patients included in the clinical trial, one patient wasexcluded after histological review and DNA was not available for twoother patients. Forty seven patients were therefore available for FCGR3Agenotype analysis. All samples were analysed in the same laboratory andDNA was extracted using standard procedures including precautions toavoid cross-contamination. DNA was isolated from peripheral blood(n=43), bone marrow (n=3) or lymph node (n=1). Genotyping ofFCGR3A-158V/F polymorphism was performed as described by Koene et al²²with a nested PCR followed by an allele-specific restriction enzymedigestion. Briefly, two FCGR3A specific primers(5′-ATATTTACAGAATGGCACAGG-3′, SEQ ID NO: 1; 5′-GACTTGGTACCCAGGTTGAA-3′,SEQ ID NO: 2) (Eurobio, Les Ulis, France) were used to amplify a 1.2 kbfragment containing the polymorphic site. The PCR assay was performedwith 1.25 μg of genomic DNA, 200 ng of each primer, 200 μmol/L of eachdNTP (MBI Fermentas, Vilnius, Lithuania) and 1 U of Taq DNA polymerase(Promega, Charbonnière, France) as recommended by the manufacturer. Thisfirst PCR consisted in 10 min at 95° C., then 35 cycles (each consistingin 3 steps at 95° C. for 1 min, 57° C. for 1.5 min, 72° C. for 1.5 min)and 8 min at 72° C. to achieve complete extension. The second PCR usedprimers (5′-ATCAGATTCGATCCTACTTCTGCAGGGGGCAT-3′ SEQ ID NO: 3;5′-ACGTGCTGAGCTTGAGTGATGGTGATGTTCAC-3′ SEQ ID NO: 4) (Eurobio)amplifying a 94 bp fragment and creating a NlaIII restriction site onlyin the FCGR3A-158V allele. This nested PCR was performed with 1 μL ofthe amplified DNA, 150 ng of each primer, 200 μmol/L of each dNTP and 1U of Taq DNA polymerase. The first cycle consisted in 5 min at 95° C.then 35 cycles (each consisting in 3 steps at 95° C. for 1 min, 64° C.for 1 min, 72° C. for 1 min) and 9.5 min at 72° C. to completeextension. The amplified DNA (10 μL) was then digested with 10 U ofNlaIII (New England Biolabs, Hitchin, England) for 12 h at 37° C. andseparated by electrophoresis on a 8% polyacrylamide gel. After stainingwith ethidium bromide, DNA bands were visualized with UV light. Forhomozygous FCGR3A-158F patients, only one undigested band (94 bp) wasvisible. Three bands (94 bp, 61 bp and 33 bp) were seen in heterozygousindividuals whereas for homozygous FCGR3A-158V patients, only twodigested bands (61 bp and 33 bp) were obtained.

FCGR2A-131H/R Genotyping

Genotyping of FCGR2A-1311-1/R was done by PCR followed by anallele-specific restriction enzyme digestion according to Liang et al²⁸.The sense primer (5′-GGAAAATCCCAGAAATTCTCGC-3′ SEQ ID NO: 5) (Eurobio)has been modified to create a BstUI restriction site in case of R allelewhereas the antisense primer (5′-CAACAGCCTGACTACCTATTACGCGGG-3′ SEQ IDNO: 6) (Eurobio) has been modified to carry a second BstUI restrictionsite that served as an internal control. PCR amplification was performedin a 50 μL reaction with 1.25 μg genomic DNA, 170 ng of each primer, 200μmol/L of each dNTP, 0.5 U of Taq DNA polymerase, and the manufacturer'sbuffer. The first cycle consisted of 3 minutes at 94° C. followed by 35cycles (each consisting in 3 steps at 94° C. for 15 seconds, 55° C. for30 seconds, 72° C. for 40 seconds) and 7 min at 72° C. to completeextension. The amplified DNA (7 μL) were then digested with 20 U ofBstUI (New England Biolabs) for 12 h at 60° C. Further analysis wasperformed as described for FCGR3A genotyping. The FCGR2A-131H and -131Ralleles were visualized as a 337 bp and 316 bp DNA fragments,respectively.

Statistical Analysis

Clinical and biological characteristics as well as clinical andmolecular responses of the patients in the different genotypic groupswere compared using a Chi-squared test or by Fisher's exact test whenappropriated. A logistic regression analysis including: sex, age (> or≦60 years), number of extra-nodal sites involved (≧ or <2), bone marrowinvolvement, BCL2 JH rearrangement status at diagnosis and FCGR3Agenotype was used to identify independent prognostic variablesinfluencing clinical and molecular responses. Progression-free survivalwas calculated according to the method of Kaplan and Meier²⁹ and wasmeasured from the start of treatment until progression/relapse or death.Comparison of the progression-free survival by FCGR3A genotype wasperformed using the log-rank test. P<0.05 was considered asstatistically significant.

Results Clinical Response

Out of the 49 patients tested for the FCGR3A-158V/F polymorphism, 10(20%) and 17 (35%) were homozygous for FCGR3A-158V and FCGR3A-158F,respectively, and 22 (45%) were heterozygous. The three groups were notdifferent in terms of sex, disease stage, bone marrow involvement,number of extra-nodal sites involved or presence of BCL2-JHrearrangement in peripheral blood and bone marrow at diagnosis (Table1). No difference was found when homozygous FCGR3A-158V patients werecompared with FCGR3A-158F carriers (FCGR3A-158F homozygous andheterozygous patients) or when homozygous FCGR3A-158F patients werecompared with FCGR3A-158V carriers (FCGR3A-158V homozygous andheterozygous patients). The objective response rate at M2 was 100%(CR+CRu=40%), 70% (CR+CRu=29%) and 64% (CR+CRu=18%) in FCGR3A-158Vhomozygous, FCGR3A-158F homozygous and heterozygous patientsrespectively (P=0.09). A significant difference in objective responserate was observed between FCGR3A-158V homozygous patients andFCGR3A-158F carriers with 67% (CR+CRu=23%) objective response rate forthis latter group (relative risk 1.5; 95% CI, 1.2-1.9; P=0.03) (Table2). No difference was observed between FCGR3A-158F homozygous patientsand FCGR3A-158V carriers. At M12, the objective response rate was 90%(CR+CRu=70%), 59% (CR+CRu=35%) and 45% (CR+CRu=32%) in FCGR3A-158Vhomozygous, FCGR3A-158F homozygous and heterozygous patientsrespectively (P=0.06). The difference in objective response rate wasstill present one year after treatment between FCGR3A-158V homozygousgroup and FCGR3A-158F carriers with 51% (CR+CRu=33%) objective responserate for this latter group (relative risk=1.7; 95% CI, 1.2-2.5; P=0.03).The logistic regression analysis showed that the homozygous FCGR3A-158Vgenotype was the only predictive factor for clinical response both at M2(P=0.02) and at M12 (P=0.01). The progression-free survival at 3 years(median follow-up: 35 months; 31-41)(FIG. 1) was 56% in FCGR3A-158Vhomozygous patients and 35% in FCGR3A-158F carriers (ns). Out of the 45patients analyzed for FCGR2A-131H/R polymorphism, 9 (20%) and 13 (29%)were homozygous for FCGR2A-131R and FCGR2A-131H, respectively, while 23(51%) were heterozygous. There was no difference in the characteristicsat inclusion or clinical response to rituximab treatment for these threegroups or for homozygous FCGR2A-131H patients and FCGR2A-131R carriers,or for homozygous FCGR2A-131R patients and FCGR2A-131H carriers (datanot shown).

Molecular Response

At diagnosis, BCL2-JH rearrangement was detected in both peripheralblood and in bone marrow in 30 (64%) patients, enabling furtherfollow-up. Twenty-five patients (six FCGR3A-158V homozygous patients and19 FCGR3A-158F carriers) and 23 patients (six FCGR3A-158V homozygouspatients and 17 FCGR3A-158F carriers) were analysed for BCL2 JHrearrangement in both peripheral blood and bone marrow at M2 and at M12(Table 3). At M2, a cleaning of BCL2-JH rearrangement was observed in3/6 of the FCGR3A-158V homozygous patients and in 5/19 of theFCGR3A-158F carriers (ns). In contrast, the rate of BCL2-JHrearrangement cleaning at M12 was higher (5/6) in the FCGR3A-158Vhomozygous patients than in the FCGR3A-158F carriers (5/17) (relativerisk=2.8; 95% CI, 1.2-6.4; P=0.03). The logistic regression analysisshowed that the FCGR3A-158V homozygous genotype was the only factorassociated with a greater probability of exhibiting BCL2 JHrearrangement cleaning at M12 (P=0.04). The single homozygousFCGR3A-158V patient still presenting with BCL2 JH rearrangement inperipheral blood and bone marrow at M12 was in CR 23 months afterrituximab treatment. In contrast, the molecular responses at M2 and M12were not influenced by the FCGR2A-131H/R polymorphism (data not shown).

DISCUSSION

Because of the increasing use of rituximab in B cell lymphoproliferativemalignancies, enhanced understanding of treatment failures and of themode of action of rituximab is required. In this regard, we genotypedFCGR3A in follicular NHL patients with well-defined clinical andlaboratory characteristics and treated with rituximab alone.⁵ Inparticular, all the patients included in this study had a low tumorburden NHL and a molecular analysis of BCL2-JH at diagnosis and duringfollow-up. The FCGR3A allele frequencies in this population were similarto those of a general caucasian population.^(23,24) Our results show anassociation between the FCGR3A genotype and the response to rituximab.Indeed, homozygous FCGR3A-158V patients, who account for one fifth ofthe population, had a greater probability of experiencing clinicalresponse, with 100% and 90% objective response rates at M2 and M12,respectively. Moreover, five of the six FCGR3A-158V homozygous patientsanalysed for BCL2-JH rearrangement showed molecular response at M12,compared to 5 of the 17 FCGR3A-158F carriers. FCGR3A-158V homozygositywas the only factor associated with the clinical and molecularresponses. However, these higher clinical and molecular responses werestill insufficient to significantly improve the progression-freesurvival in homozygous FCGR3A-158V patients.

This is the first report of an easily assessable genetic predictivefactor for both clinical and molecular responses to rituximab. However,the genetic association does not demonstrate the mode of action ofrituximab involves FcγRIIIa. The association observed between FCGR3Agenotype and response to rituximab might be due to another geneticpolymorphism in linkage disequilibrium. Those polymorphisms could belocated in FCGR3A itself like the triallelic FCGR3A-48L/H/Rpolymorphism³¹ or in other FcγR-coding genes, since FCGR3A is located onthe long arm of chromosome 1, which includes the three FCGR2 genes andFCGR3B.³² A linkage disequilibrium has been reported between FCGR2A andFCGR3B.³³ However, the fact that FCGR2A-131H/R polymorphism was notassociated with a better response to rituximab strongly supports thefact that a gene very close to FCGR3A or FCGR3A itself is directlyinvolved.

Several in vitro studies argue in favor of direct involvement ofFCGR3A-158V/F polymorphism. First, Koene et a/²³ have shown that thepreviously reported differences in IgG binding among the threeFcγRIIIa-48L/H/R isoforms³¹ are a consequence of the linkedFcγRIIIa-158V/F polymorphism and several teams have demonstrated that NKcells from individuals homozygous for the FCGR3A-158V allotype have ahigher affinity for human complexed IgG1 and are more cytotoxic towardsIgG1-sensitized targets ^(23,24,34) Our present results establish thatFCGR3A-158V homozygous patients have a better response to rituximab,which is probably due to a better in vivo binding of that chimeric humanIgG1 to FcγRIIIa. Secondly, NK cell- and macrophage-mediated ADCC is oneof the mechanisms triggered by anti-CD20 antibodies in vitro^(8,11,12)as well as in murine models in vivo, ¹⁷⁻¹⁹ and rituximab-mediatedapoptosis is amplified by FcγR-expressing cells.^(15,16) Out of allFcγR, FcγRIIIa is the only receptor shared by NK cells and macrophages.We thus postulate that FCGR3A-158V patients show a better response torituximab because they have better ADCC activity against lymphoma cells.The fact that more than 50% of the FCGR3A-158F carriers nonethelesspresent a clinical response to rituximab could be explained by lower,but still sufficient, ADCC activity or, more likely, by other mechanismsoperating in vivo such as complement-dependent cytotoxicity,complement-dependent cell-mediated cytotoxicity^(11,13,14) and/orapoptosis.^(15,16) ADCC could then be viewed as an additional mechanismin the response to rituximab that is particularly effective inFCGR3A-158V homozygous patients.

The in vitro studies suggest a “gene-dose” effect with a level of IgG1binding to NK cells from FCGR3A heterozygous donors intermediate betweenthat observed with NK cells from FCGR3A-158V and FCGR3A-158Fhomozygotes²³. However, the clinical response of heterozygous patientsappears similar to that of FCGR3A-158F homozygous patients. Furtherstudies with larger groups of patients will be required to concludeagainst a “gene-dose” effect in vivo.

Since FcγRIIIa is strongly associated with a better response torituximab, it needs to be taken into account in the development of newdrugs targetting the CD20 antigen. For example, it may be possible touse engineered rituximab to treat FCGR3A-158F-carrier patients with Bcell lymphomas. Indeed, by modifying various residues in the IgG1 lowerhinge region, Shields et al have recently obtained IgG1 mutants whichbind more strongly to FcγRIIIa-158F than native IgG1³⁴.

Taken together, these results allow to set up new therapeutic strategiesagainst B lymphoproliferative disorders based upon prior determinationof the patients FCGR3A genotype. Since this polymorphism has the samedistribution in various ethnic population, including blacks andJapanese, such a strategy may be applied worldwide.^(23,35,36)Furthermore, such a pharmacogenetic approach may also be applied toother intact humanized IgG1 antibodies used in the treatment of B cellmalignancies, such as Campath-1H, or those used in the treatment ofother malignancies, such as trastuzumab (Herceptin®). Even moregenerally, this approach may apply to other intact (humanized)therapeutic (IgG1) antibodies developed to deplete target cells.

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TABLE 1 CHARACTERISTICS OF PATIENTS ACCORDING TO THE FCGR3A-158V/FPOLYMORPHISM. FCGR3A- FCGR3A- FCGR3A- 158VV 158VF 158FF p* n (%) 10(20%) 22 (45%) 17 (35%) Sex M 3 12 10 ns F 7 10 7 Disease stage II-III 36 6 ns IV 7 16 11 Bone marrow involvement yes 7 16 9 ns no 3 6 8Extra-nodal sites involved <2 8 20 13 ns ≧2 2 2 4 BCL2-JH rearrangement8 12 11 ns in peripheral blood BCL2-JH rearrangement 7 12 11 ns in bonemarrow *Satistical comparisons of the three groups of homozygousFCGR3A-158V patients vs FCGR3A-158F carriers and of homozygousFCGRA-158F patients against FCGR3A/158V carriers.

TABLE 2 CLINICAL RESPONSE TO RITUXIMAB BY FCGR3A-158V/F POLYMORPHISM.FCGR3A-158F FCGR3A-158VV carriers p* Clinical response at M2 Objectiveresponse 10 (100%) 26 (67%) 0.03 complete remission 3 7 completeremission 1 2 unconfirmed partial response 6 17 No response 0 (0%) 13(33%) no change 0 10 progressive disease 0 3 Clinical response at M12Objective response 9 (90%) 20 (51%) 0.03 complete remission 6 11complete remission 1 2 unconfirmed partial response 2 7 No response 1(10%) 19 (49%) no change 0 2 progressive disease 1 17 *Satisticalcomparison of homozygous FCGR3A-158V patients against FCGR3A-158Fcarriers. Data concerning the three genotype subgroups are given in thetext.

TABLE 3 MOLECULAR RESPONSE TO RITUXIMAB AT M2 AND AT M12 BY THEFCGR3A-158V/F POLYMORPHISM. FCGR3A-158F FCGR3A-158VV carriers pMolecular response at M2 ns Cleaning of BCL2-JH 3 5 rearrangementPersistent BCL2-JH 3 14 rearrangement Molecular response at M12 0.03Cleaning of BCL2-JH 5 5 rearrangement Persistent BCL2-JH 1 12rearrangement

1. A method of assessing the response of a subject to a therapeuticantibody treatment, comprising determining in vitro the FCGR3A158genotype of said subject.
 2. A method of selecting patients fortherapeutic antibody treatment, the method comprising determining invitro the FCGR3A158 genotype of said subject.
 3. A method of improvingthe efficacy or treatment condition or protocol of a therapeuticantibody treatment in a subject, comprising determining in vitro theFCGR3A158 genotype of said subject.
 4. The method of claim 1, comprisingdetermining amino acid residue at position 158 of FcγRIIIa receptor, aValine at position 158 being indicative of a better response to saidtreatment and a phenylalanine at position 158 being indicative of alower response to said treatment.
 5. The method of claim 1, whereindetermining amino acid residue at position 158 of FcγRIIIa receptorcomprises a step of sequencing the FcγRIIIa receptor gene or RNA or aportion thereof comprising the nucleotides encoding amino acid residue158.
 6. The method of claim 1, wherein determining amino acid residue atposition 158 of FcγRIIIa receptor comprises a step of amplifying theFcγRIIIa receptor gene or RNA or a portion thereof comprising thenucleotides encoding amino acid residue
 158. 7. The method of claim 6,wherein amplification is performed by polymerase chain reaction (PCR),such as PCR, RT-PCR and nested PCR.
 8. The method of claim 1, whereindetermining amino acid residue at position 158 of FcγRIIIa receptorcomprises a step of allele-specific restriction enzyme digestion.
 9. Themethod of claim 1, wherein determining amino acid residue at position158 of FcγRIIIa receptor comprises a step of hybridization of theFcγRIIIa receptor gene or RNA or a portion thereof comprising thenucleotides encoding amino acid residue 158, with a nucleic acid probespecific for the genotype Valine or Phenylalanine.
 10. The method ofclaim 1, wherein determining amino acid residue at position 158 ofFcγRIIIa receptor comprises: Obtaining genomic DNA from a biologicalsample, Amplifying the FcγRIIIa receptor gene or a portion thereofcomprising the nucleotides encoding amino acid residue 158, anddetermining amino acid residue at position 158 of said FcγRIIIa receptorgene.
 11. The method of claim 1, wherein determining amino acid residueat position 158 of FcγRIIIa receptor comprises: Obtaining genomic DNAfrom a biological sample, Amplifying the FcγRIIIa receptor gene or aportion thereof comprising the nucleotides encoding amino acid residue158, Introducing an allele-specific restriction site, Digesting thenucleic acids with the enzyme specific for said restriction site and,Analysing the digestion products, i.e., by electrophoresis, the presenceof digestion products being indicative of the presence of the allele.12. The method of claim 1, wherein determining amino acid residue atposition 158 of FcγRIIIa receptor comprises: total (or messenger) RNAextraction from cell or biological sample or biological fluid in vitroor ex vivo, optionally cDNA synthesis, (PCR) amplification with specificFCGRIIIa oligonucleotide primers, and analysis of PCR products.
 13. Themethod of claim 1, wherein determining amino acid residue at position158 of FcγRIIIa receptor comprises a step of sequencing the FcγRIIIareceptor polypeptide or a portion thereof comprising amino acid residue158.
 14. The method. of claim 1, wherein the subject is a human subject.15. The method of claim 14, wherein the subject has a tumor, a viralinfection, or a disease condition associated with allogenic orpathological immunocompetent cells.
 16. The method of claim 15, whereinthe subject has a tumor and the therapeutic antibody treatment aims atreducing the tumor burden.
 17. The method of claim 16, wherein the tumoris a lymphoma, particularly a NHL.
 18. The method of claim 1, whereinthe antibody is an IgG1 or an IgG3.
 19. The method of claim 18, whereinthe antibody is an anti-CD20 antibody, particularly rituximab.